Structural characterization of enterobactin hydrolase IroE

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Pathogenic bacteria require iron for proliferation, often limited by environmental scarcity.
• Bacteria utilize siderophores to chelate and import iron, a critical metabolic process.
• Serine hydrolases Fes, IroD, and IroE are key enzymes in bacterial iron scavenging pathways.

Purpose of the Study:

• To determine the crystal structure of the IroE enzyme from uropathogenic Escherichia coli CFT073.
• To elucidate the structural basis of IroE's catalytic mechanism and compare it to related enzymes.

Main Methods:

• X-ray crystallography was used to determine the native structure of IroE and its complex with diisopropyl fluorophosphonate (DFP).
• Site-directed mutagenesis was employed to investigate the roles of key active site residues.
• Structural comparison was performed between IroE and the Fes enzyme from Shigella flexneri.

Main Results:

• The crystal structure of IroE revealed a typical alpha/beta-hydrolase fold with an atypical catalytic dyad (Ser189, His287).
• An atypical oxyanion hole involving Arg130, anchored by Asp90, was identified and found essential for catalysis.
• Comparison with Fes highlighted structural differences, including an amino-terminal lid domain in Fes, potentially conferring specificity.

Conclusions:

• The structure of IroE provides insights into the catalytic mechanism of bacterial serine hydrolases involved in iron acquisition.
• The identified atypical active site features in IroE are critical for its enzymatic activity.
• Structural variations, like the lid domain in Fes, may explain functional specificity among related hydrolases.